ZRANB2 localizes to supraspliceosomes and influencesthe alternative splicing of multiple genes in the transcriptome

Yee Hwa J. Yang • M. Andrea Markus •

A. Helena Mangs • Oleg Raitskin • Ruth Sperling •

Brian J. Morris

Received: 5 October 2012 / Accepted: 1 May 2013 / Published online: 11 May 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Alternative splicing is a major source of protein

diversity in humans. The human splicing factor zinc finger,

Ran-binding domain containing protein 2 (ZRANB2) is a

splicing protein whose specific endogenous targets are

unknown. Its upregulation in grade III ovarian serous

papillary carcinoma could suggest a role in some cancers.

To determine whether ZRANB2 is part of the supras-

pliceosome, nuclear supernatants from human embryonic

kidney 293 cells were prepared and then fractioned on a

glycerol gradient, followed by Western blotting. The same

was done after treatment with a tyrosine kinase to induce

phosphorylation. This showed for the first time that

ZRANB2 is part of the supraspliceosome, and that phos-

phorylation affects its subcellular location. Studies were

then performed to understand the splicing targets of

ZRANB2 at the whole-transcriptome level. HeLa cells

were transfected with a vector containing ZRANB2 or with

a vector-only control. RNA was extracted, converted to

cDNA and hybridized to Affymetrix GeneChip� Human

Exon 1.0 ST Arrays. At the FDR B1.3 significance level

we found that ZRANB2 influenced the alternative splicing

of primary transcripts of CENTB1, WDR78, C10orf18,

CABP4, SMARCC2, SPATA13, OR4C6, ZNF263,

CAPN10, SALL1, ST18 and ZP2. Several of these have

been implicated in tumor development. In conclusion

ZRANB2 is part of the supraspliceosome and causes dif-

ferential splicing of numerous primary transcripts, some of

which might have a role in cancer.

Keywords Cancer � Pre-mRNA splicing � Spliceosome �ZNF265 � Zinc finger

Introduction

Alternative splicing is a major source of protein diversity in

eukaryotic organisms and is often regulated in a tissue or

developmental stage-specific manner. Genome-wide stud-

ies have shown that in humans the primary RNA transcriptElectronic supplementary material The online version of thisarticle (doi:10.1007/s11033-013-2637-9) contains supplementarymaterial, which is available to authorized users.

Y. H. J. Yang

School of Mathematics and Statistics, The University of Sydney,

Sydney, NSW 2006, Australia

M. A. Markus � A. H. Mangs � B. J. Morris (&)

Basic & Clinical Genomics Laboratory, School of Medical

Sciences and Bosch Institute, University of Sydney, Building

F13, Sydney, NSW 2006, Australia

e-mail: brian.morris@sydney.edu.au

Present Address:

M. A. Markus

Department of Haematology and Oncology, Centre for Internal

Medicine, University Medical Centre Goettingen,

37075 Goettingen, Germany

Present Address:

A. H. Mangs

Ramaciotti Centre for Gene Function Analysis, University of

New South Wales, DO6, Sydney, NSW 2052, Australia

O. Raitskin � R. Sperling

Department of Genetics, The Hebrew University of Jerusalem,

Jerusalem, Israel

Present Address:

O. Raitskin

Department of Cell & Developmental Biology, John Innes

Centre, Norwich Research Park, Norwich NR4 7UH, UK

123

Mol Biol Rep (2013) 40:5381–5395

DOI 10.1007/s11033-013-2637-9

of 95 % of genes can undergo alternative splicing [1, 2], so

explaining the large number of proteins that are generated

from a much smaller number of genes. Alternative splicing

of pre-mRNA transcripts can greatly affect the function,

localization, binding properties and activity of a protein [3].

The human splicing factor zinc finger, Ran-binding

domain containing protein 2 (ZRANB2) was identified

originally in a differential display experiment involving

2-day and 10-day primary cultures of rat juxtaglomerular

cells [4]. During prolonged culture Zranb2 mRNA was

found to undergo downregulation in concert with renin

mRNA, the archetypical constituent of these cells [4]. We

then cloned the mouse and human homologs [5]. ZRANB2

has two zinc finger domains, a glutamic acid-rich region

and a C-terminal Ser/Arg-rich (SR) domain and as such

resembles members of the SR-related family of splicing

factors [6]. The two N-terminal zinc fingers (ZnFs) show

striking homology to RanBP2 Ran-binding protein

domains. These ZnFs were shown to recognize single-

stranded (ss) RNA with high affinity and specificity, sug-

gesting that the regulation of alternative splicing by

ZRANB2 is via a direct interaction with pre-mRNA at sites

that resemble the consensus 50 splice site [7]. Orthologs of

ZRANB2 exist across phyla, examples being TAF15 in

Aridopsis thaliane, Y25C1A.8 in Caenorhabditis elegans,

CG3732 in Drosophila melanogaster, wufa96h03 in Danio

rerio, XI.520 in Xenopus laevis, ZRANB2 in Anolis caro-

linensis, ZRANB2 in Gallus gallus and ZRANB2 in Mus

musculus [8]. The RNA recognition motif of the zinc fin-

gers in particular are highly conserved throughout evolu-

tion. This degree of similarity is consistent with an

important role of the RNA-binding activity and splicing

function of ZRANB2 [7].

As well as binding to pre-mRNA, ZRANB2 also binds

to the essential splicing factors U170 K, U2AF and

SFRS17A (formerly known as XE7) [6, 9]. We, and others,

have shown that ZRANB2 alters the splicing patterns of

primary transcripts of the minigenes CD44, Tra2b1,

SRSF3, GluR-B and SMN2, doing so in a dose-dependent

manner [6, 9, 10]. At this stage there is no evidence to

exclude the possibility that ZRANB2 is part of the core

spliceosome machinery.

The role of ZRANB2 in the regulation of alternative

splicing globallyis, however, still unclear. To determine the

endogenous transcripts that are targeted by ZRANB2 at the

transcriptome-wide level, we performed exon microarray

studies to determine differences in splicing in human cells

overexpressing ZRANB2. We also ascertained whether

ZRANB2 might be a component of the supraspliceosome,

which is a subcellular structure in which endogenous pre-

mRNAs are packaged with all five spliceosomal U

snRNPs, together with other splicing factors and additional

pre-mRNA processing factors [11, 12].

Materials and methods

Supraspliceosome experiment

Supraspliceosomes were prepared from human embryonic

kidney (HEK) 293 cells using a protocol described previ-

ously [13]. Briefly, nuclear supernatants enriched for su-

praspliceosomes were prepared from purified nuclei of

HEK293 cells by micro-sonication of the nuclei and pre-

cipitation of the chromatin in the presence of tRNA. The

nuclear supernatants were fractionated in 10–45 % glycerol

gradients containing 100 mM NaCl, 10 mM Tris�HCl, pH

8.0, 2 mM MgCl2 and 2 mM vanadyl ribonucleoside com-

plex. Centrifugations were carried out at 4 �C in a SW41

rotor run at 288,000 relative centrifugal force (rcf) for

90 min. The gradients were calibrated with 200S tobacco

mosaic virus (TMV) particles run in a parallel gradient

peaking in fractions 10 and 11. It has been shown previously

that supraspliceosomes sediment in fractions 8–14 with a

peak corresponding to fractions 10 and 11, as confirmed by

electron microscopic visualization and sedimentation of the

hnRNP G protein, which is associated with supraspliceo-

somes, and sedimentation at 200S serves as an unambiguous

marker for these large ribonucleoprotein complexes [14].

hnRNP G was detected on Western blots using a specific

antibody. The distribution of ZRANB2 across the gradient

was analyzed by Western blotting using a polyclonal anti-

body to ZRANB2 (see ‘‘Antibodies’’ section below.)

Constructs

GFP-ZRANB2 was cloned as described previously [9]. We

have shown previously that human ZRANB2 pre-mRNA is

alternatively spliced to give two variants with different 30

ends, and that both isoforms are present in the nucleus of

human cells [6, 9]. The construct containing isoform 2

(short form, SF) of ZRANB2 was used. This construct

included exon 1 through to the alternatively spliced exon

10. Isoform 2 was chosen since it has been shown previ-

ously to affect alternative splicing of a Tra2b1 minigene

[6]. An expression construct, CMV-CSK, encoding C-ter-

minal Src tyrosine kinase (CSK) was provided by Stefan

Stamm, University of Kentucky.

Antibodies

Polyclonal ZRANB2 antibody was generated to order by

Alpha Diagnostics, San Antonio, TX by injection into

rabbits of two peptides whose sequences were

STKNFRVSDG and KTLAEKSRGLFSA, each of which

are present in both the short and long forms of ZRANB2.

The hnRNP G antibody was provided by Stefan Stamm,

University of Kentucky.

5382 Mol Biol Rep (2013) 40:5381–5395

123

Cell culture and transfection

Human cervical cancer (HeLa) cells were obtained from

the American Type Culture Collection and cultured at

37 �C under 5 % CO2 in Dulbecco’s Modified Eagle’s

Medium supplemented with 0.1 mM non-essential amino

acids, 1.0 mM sodium pyruvate, 10 % fetal bovine serum

and 5,000 units/mL penicillin/streptomycin (all from

Invitrogen). Constructs were transfected into cells with

Lipofectamine 2000 (Invitrogen, Australia) according to

the manufacturer’s instructions. Five flasks of cells were

transfected with 300 lg of GFP-ZRANB2 plasmid, five

were transfected with GFP plasmid control, and five were

left untransfected. Cells were harvested after 40 h of cul-

ture and RNA was extracted using RNeasy RNA extraction

kit (Qiagen, Australia). RNA was assessed for quality

based on an RNA integrity number (RIN) higher than 8 by

electrophoresis involving an Agilent 2100 Bioanalyzer at

the Ramaciotti Centre for Gene Function Analysis, Uni-

versity of New South Wales, Sydney, Australia. RNA was

quantified by spectrophotometry (NanoDrop� ND-100

spectrophotometer, Thermo Scientific, USA).

Exon arrays

One microgram of each RNA sample was processed using

the Affymetrix GeneChip Whole Transcript Sense Target

Labeling Assay (Affymetrix). Briefly, control poly-A RNA

was added to 1 lg RNA, followed by a ribosomal RNA

reduction step (RiboMinus step). After determining RNA

concentration, we performed first cycle cDNA synthesis

and clean-up of antisense cRNA, followed by second cycle

cDNA synthesis and then cRNA hydrolysis. DNA was then

fragmented and labeled terminally for hybridization to the

array. One microarray was performed for each sample,

with no pooling. Samples were hybridized to Affymetrix

GeneChip� Human Exon 1.0 ST Arrays, each of which

contain approximately 5.4 million probes grouped into 1.4

million probe sets. These probe sets span over 1 million

exon clusters, that is, exon annotations from various

sources that overlap by genomic locations. Thus, exon

arrays cover the whole transcriptome, in comparison to 30

tailing arrays that only target the 30 end of each transcript.

Exon arrays are high density arrays at 5 lm feature size.

Affymetrix exon arrays correspond well to standard

expression arrays [15, 16]. The array steps were carried out

according to the manufacturer’s instructions, and with the

assistance of the Ramaciotti Centre who performed the

hybridization of cDNA to the array, as well as washing and

scanning of the arrays. The exon array permits analysis of

the level of expression of individual exons, which were

measured. Specific alternative splice junctions are not

detected because array oligonucleotides do not span exon

junctions, instead being situated within exons, meaning

that no information could be provided about exon junc-

tions. The data set obtained has been deposited in the NCBI

Gene Expression Omnibus database according to MIAME

guidelines with accession numbers GSE32932.

Statistical analysis of array data

Results from the Affymetrix exon arrays were background-

corrected, quantile normalized and summarized at the exon

and whole transcript level using the robust multi-array

average (RMA) algorithm [17] implemented in the Affyme-

trix Power Tools software package [18]. Quality assessment

indicated an outlier among the ZRANB2 replicates and this

replicate was removed from further analysis. The probe

annotation package ‘‘hugene10sttranscriptcluster.db’’ from

Bioconductor [19] was used. A two-stage approach was used

to identify differentially expressed exons of transcripts. The

first stage was to identify differentially expressed exons

between the two groups, i.e., ZRANB2 and GFP-only control.

For a typical transcript ‘‘g’’ (log-likelihood ratio) we aver-

aged expression values among all exons within transcript ‘‘g’’

and computed the fold-change and moderated t-statistics [20]

between groups. We then generated a candidate list of dif-

ferentially expressed exons with an absolute moderated t-

statistic of C2, and that showed a fold change of[1.5-fold

between groups. These procedures involved a robust least

squares approach implemented in the Limma library of the

Bioconductor software package [21]. The first stage identi-

fied 681 potential differentially expressed transcripts from

5,563 exons. The second stage was to identify exon level

differences within differentially expressed transcripts. For

each exon, we computed fold-change and log-odds ratios and

generated a ranked list of differentially expressed exons,

where an exon with a large log-odds ratio was assumed to be

more likely to be differentially expressed than not. This

analysis was complemented by one using the Partek

Genomics Suite Software (Partek Inc.).

qPCR

We chose several of the leading candidates from the exon

arrays for verification. RNA was extracted from cells and

used for semi-quantitative reverse transcriptase real-time

PCR using primers in the exons flanking an exon of

interest. For SMARCC2 the following primers were used:

50-cctacctctcacttccatgtcttg-30 (F, exon 17) and 50-tgtgtaca

tgtctgtgcgcag-30 (R, exon 19 variant 2); for WDR78:

50-tgaacctgaagagcctgaagatg-30 (F, exon 9) and 50-ccagaa

ctggaacattactgttgct-30 (R, exon 11); and for SPATA13 50-gt

tcctgctcacaccagtgc-30 (F, exon 8) and 50-aagaacgtccgct

gctgg-30 (R, exon 10). The PCR program used for

SMARCC2 and WDR78 was: 23 or 30 cycles of 95 �C for

Mol Biol Rep (2013) 40:5381–5395 5383

123

30 s, 59 �C for 30 s and 72 �C for 30 s. For SPATA13 the

program used was 30 cycles of 95 �C for 30 s, 60 �C for

30 s and 72 �C for 30 s. The band intensities were quan-

tified using Image J (Java).

Results

ZRANB2 localizes to supraspliceosomes

Because ZRANB2 was shown to affect splicing, we asked

whether ZRANB2 is part of the supraspliceosome, a

macromolecular machine active in splicing and in which

the entire repertoire of nuclear pre-mRNAs are individually

packaged [11]. Supraspliceosomes harbor all known

splicing factors; in particular, the phosphorylated forms of

the SR protein family and the regulatory splicing factor

hnRNP G are predominantly associated with supras-

pliceosomes so making them unambiguous markers

[11, 14]. When nuclear supernatants enriched for supras-

pliceosomes were fractionated in glycerol gradients,

endogenous ZRANB2 was found sedimenting in fractions

13–16, as analyzed by Western blotting using a polyclonal

anti-ZRANB2 antibody (Fig. 1a, upper panel). However,

as can be seen in Fig. 1a, lower panel, after inducing

intracellular protein phosphorylation of the cells with the

tyrosine kinase CSK, ZRANB2 was found localized in

heavier fractions (8–16, with a peak in fractions 12 and 13).

Figure 1b shows that induction of phosphorylation caused

ZRANB2 to co-sediment with the regulatory splicing fac-

tor hnRNP G which was previously shown to be predom-

inantly associated with supraspliceosomes [14].

ZRANB2 overexpression influences alternative splicing

of multiple primary transcripts

Over 90 % of cells were successfully transfected with

GFP-ZRANB2 and GFP-only constructs (Fig. 2). Initial

comparison of array results for untransfected cells with

cells transfected with the GFP-only plasmid showed some

influences of the latter on exon expression (data not

shown). We therefore deemed the GFP-only transfected

cells as being a more suitable control than the untransfected

cells. Differences in exon expression between GFP-

ZRANB2 and GFP-only were thereby taken as indicative

of effects of ZRANB2 on alternative splicing. Table 1

shows that based on an alternative splice statistic of t [ 2

and false discovery rate (FDR) of B1.3 there were 12

transcripts that underwent alternative splicing of one or

more of their exons in response to ZRANB2 overexpres-

sion in HeLa cells. The number of exonic regions that each

transcript possesses are shown, together with the ID num-

bers of those exons that were alternatively spliced in

response to ZRANB2 overexpression. Figure 3 illustrates

the effect of ZRANB2 on splice site selection of these 12

pre-mRNAs. A difference in expression was taken as

indicative of inclusion (up-regulation) or exclusion (down-

regulation) of a particular exon. As an example, ZRANB2

overexpression favored the expression of a splice isoform

of the WD repeat domain 78 (WDR78) transcript lacking

exon 10 (Fig. 3b). This was confirmed by qPCR of exon 10

which showed a twofold difference in DCT values for

ZRANB2 samples compared to GFP control (P = 0.026).

In the case of spermatogenesis associated 13 (SPATA13)

pre-mRNA, the array data showed that overexpression of

ZRANB2 increased an isoform that included exon 9

(Fig. 3f), but this was not confirmed by qPCR, which

yielded only one band (data not shown). Another primary

transcript that ZRANB2 affected the alternative splicing of

was that of SWI/SNF related, matrix associated, actin

dependent regulator of chromatin, subfamily c, member 2

(SMARCC2) (Fig. 3e). ZRANB2 caused exon 18 sequen-

ces to be excluded from the primary SMARCC2 transcript,

resulting in a protein isoform lacking 31 amino acids and

having a different C-terminus.

The top 62 primary transcript targets of ZRANB2 gen-

erated from the arrays (FDR B0.98) are provided in

(Supplementary Table 1).

Discussion

The present study is the first to describe ZRANB2 as being

part of the supraspliceosome and to show that its overex-

pression affects alternative splicing of specific transcripts

at the genome-wide level. Our study also showed that

phosphorylation increased expression of ZRANB2 in

supraspliceosomes.

It is well known that phosphorylation of splicing factors

such as SR proteins and heterogeneous ribonucleoprotein

(hnRNP) particles can affect both their activity and cellular

localization. For example dephosphorylation regulates the

shuttling of ASF/SF2, as well as preventing the non-shut-

tling of SC35, between the cytoplasm and the nucleus [22].

Moreover, SRPK1, by phosphorylating the N-terminal RS

domain of ASF/SF2 in the cytoplasm, mediates the nuclear

import of ASF/SF2, as well as its recruitment to nuclear

speckles [23]. In addition, phosphorylation of polypyrimi-

dine tract binding (PTB)-associated splicing factor (PSF)

inhibits PSF binding to the 30 polypyrimidine tract of pre-

mRNA, so providing further evidence of the functional

importance of phosphorylation [24]. The functional sig-

nificance of ZRANB2 in supraspliceosomes in response to

stimulation of phosphorylation in cells requires further

investigation.

5384 Mol Biol Rep (2013) 40:5381–5395

123

In mice, it has been reported that the overexpression of

isoform 2 of ZRANB2 decreases the expression of isoform

1, suggesting that the alternative splicing of Zranb2 pre-

mRNA is self-regulated [25]. Since these results were

generated using overexpressed isoform 2, it is possible that

the decrease in isoform 1 compared to isoform 2 was an

Fig. 1 ZRANB2 is found in the supraspliceosome. a, b, Nuclear

supernatants enriched in supraspliceosomes were prepared from

HEK293T cells, either untreated (upper panels), or treated with the

protein-tyrosine kinase CSK to stimulate phosphorylation (lower

panels). The nuclear supernatants were fractionated in a 10–45 %

glycerol gradient and effluent was collected from bottom to top in 20

fractions. Supraspliceosomes sediment at fractions 8–14. The gradi-

ents were calibrated with 200S TMV particles run in a parallel

gradient peaking in fractions 10 and 11. a, Western blot analysis

performed with anti-ZRNAB2 antibodies on untreated cells (upper

panel) and CSK treated cells (lower panel). b, Western blot analysis

with anti-hnRNP G antibodies on untreated cells (upper panel) and

CSK treated cells (lower panel). hnRNP G was previously shown to

be associated with supraspliceosomes [14]. ?CSK, treatment of cells

with the protein-tyrosinase CSK

Fig. 2 HeLa cells after transfection with GFP-ZRANB2 (a) and

GFP-only (b) plasmids. The green fluorescence apparent in these

representative sections indicated a predominantly nuclear localization

of the GFP-ZRANB2 plasmid and that over 90 % of the cells had

been transfected, while the GFP-only control was cytoplasmic as

expected

Mol Biol Rep (2013) 40:5381–5395 5385

123

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5386 Mol Biol Rep (2013) 40:5381–5395

123

Fig. 3 Bar graphs showing the most significant results from the exon

array experiments. Each panel shows a primary transcript affected by

ZRANB2 overexpression in HeLa cells. Individual exons of each

primary transcript that were differentially expressed are indicated by

significant difference between GFP-ZRANB2 over-expressing sam-

ples (n = 5) versus GFP vector-only control (n = 5). The plots were

generated in R using the package ggplot2. Note that the scale on the

ordinate is logarithmic (log2). a CENTB1, b WDR78 (exon 2417027

downregulated threefold), c C10orf18, d CABP4, e SMARCC2,

f SPATA13 (exon 3451591 upregulated tenfold), g OR4C6,

h ZNF263, i CAPN10, j SALL1, k ST18, l ZP2

Mol Biol Rep (2013) 40:5381–5395 5387

123

Fig. 3 continued

5388 Mol Biol Rep (2013) 40:5381–5395

123

Fig. 3 continued

Mol Biol Rep (2013) 40:5381–5395 5389

123

Fig. 3 continued

5390 Mol Biol Rep (2013) 40:5381–5395

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Fig. 3 continued

Mol Biol Rep (2013) 40:5381–5395 5391

123

Fig. 3 continued

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effect of overexpression. Certainly, in the present study, we

did not see any evidence that ZRANB2 autoregulates its

two own alternatively spliced isoforms, since we failed to

observe a decrease in exon 10 of isoform 1 in response to

ZRANB2 overexpression. We chose to use isoform 2 of

ZRANB2 for the present experiments, since a previous

study by us showed that isoform 2 affects the alternative

splicing of a Tra2b1 minigene [6]. Despite this, the pos-

sibility remains that isoform 1 might be the major

ZRANB2 isoform involved in regulation of splicing and

that isoform 2 has only a minor role in such regulation. The

latter possibility has been noted in the mouse, in that iso-

form 1 is the major ZRANB2 isoform and regulates the

alternative splicing of GluR-B and Smn2 pre-mRNAs [10].

Our exon array data suggest that the major transcript

targets of ZRANB2 include the pre-mRNAs of WDR78,

SPATA13 and SMARCC2. WDR78 is a novel protein with a

WD40 domain that is found in a number of eukaryotic

proteins having a wide variety of functions, including

participation as adaptor/regulatory modules in signal

transduction, pre-mRNA processing and cytoskeleton

assembly [26, 27]. Two isoforms have been reported pre-

viously for WDR78, one of which is 848 amino acids

longer than the other. One of its transcripts uses a different

splice site in the 30 coding region. This leads to an isoform

having a shorter and distinct C-terminus. We showed that

ZRANB2 affects the alternative splicing of the WDR78

primary transcript so as to favor a form of WDR78 lacking

exon 10. However, the two isoforms of WDR78 that have

been reported previously both contain exon 10. The func-

tional significance of the effect ZRANB2 on WDR78 iso-

forms remains to be determined.

SPATA13 (also known as ASEF2) is required for cell

migration, including that of colorectal tumor cells

expressing truncated APC [28] and hepatocyte growth

factor-induced cell migration that regulates actin cyto-

skeletal organization [29]. APC is tumor suppressor

mutated in sporadic and familial colorectal cancers [30].

The promotion of cell migration and rapid adhesion turn-

over appears to be controlled by the regulation of the

activities of the Rho-family of GTPases [31]. SPAT13

(ASEF2) shows significant structural and functional simi-

larities to ASEF1. Both of these Rho family guanine

nucleotide exchange factors are CDC42 exchange factors

[32].

The protein encoded by SMARCC2 is a member of the

SWI/SNF (mating-type switch/sucrose nonfermenting)

family of proteins, whose members display helicase and

ATPase activities. The SWI/SNF complex is involved in

chromatin remodeling on promoters, but has also been

detected on the coding region of genes. The SMARCC2

protein contains a leucine zipper motif typical of many

transcription factors. Transfection of SMARCC2 decreases

cell viability in vitro [33]. The chicken homolog of

SMARCC2 was found in a Gallus gallus supraspliceosome

extract [34]. Interestingly, SWI/SNF can function as a

regulator of splicing through its catalytic subunit Brm [35].

Brm also associates with U1 and U5 small nuclear RNAs

(snRNAs), as well as PRP6 and Sam68 proteins, implying

that it is involved in recruitment of the splicing machinery

[35]. It thus appears that ZRANB2 may be able to regulate

other splicing regulators. Splicing factors have been shown

previously to regulate the splicing of other splicing factors.

For example, ASF/SF2 has been shown to antagonize the

auto-regulatory activity of SRSF3, by suppressing the

production of an exon 4-included form of SRSF3 [36].

Similarly, ASF/SF2, SRp30c, SAF-B and hnRNP G influ-

ence splicing of the human SR-like protein Tra2b1 by

excluding exon 2 and thereby decreasing the production of

the b4 form of Tra2b1 [37].

Changes in concentration, localization or activity of

splicing factors have been shown previously to affect the

process of splicing and to give rise to protein isoforms with

tumorigenic properties [38]. SMARCC2 has been mapped

to a chromosomal region that is frequently involved in

human cancers [39]. SMARCC2 is, moreover, overex-

pressed in squamous non-small cell lung cancers in com-

parison to normal bronchial epithelial tissue [40]. As

mentioned above, it has been shown that SPATA13 is

required for migration of colorectal tumor cells expressing

truncated APC [28]. Since ZRANB2 affects the alternative

splice site selection of SMARCC2 and SPATA13 pre-

mRNAs, we speculate that ZRANB2 might be involved in

cancer. Of interest, a DNA microarray experiment to

characterize the global expression pattern in surface epi-

thelial cancers of the ovary showed that ZRANB2 is highly

expression in grade III ovarian serous papillary carcinomas

[41]. Our data therefore add to previous findings using

exon arrays that have found alternative splicing events in

several cancer models [42, 43].

A limitation of the present work was the use of a GFP-

containing vector, in that cells transfected with this par-

ticular vector-only control showed a large number of

changes in alternative splicing. The use of GFP was,

however, necessary in order to visualize the transfection

efficiency. The approach of next generation sequencing

could be used to confirm the present findings. This would

overcome any array system bias towards representing

cassette exons, especially if as a previous study suggests

ZRANB2 might be involved in 50 splice site selection [7].

In conclusion, we have shown that ZRANB2 is part of

the supraspliceosome and is able to regulate the alternative

splicing of a number of physiological transcripts from the

genome. A role of ZRANB2 in cancer and tumor pro-

gression suggested by our findings will require confirma-

tion by future experiments.

Mol Biol Rep (2013) 40:5381–5395 5393

123

Acknowledgments We thank Stefan Stamm, University of Ken-

tucky Chandler Medical Center, for the CMV-CSK construct and for

the hnRNP G antibody used in the supraspliceosome experiments. We

also thank Dr. Helen Speirs at the Ramaciotti Centre for Gene

Function Analysis, University of New South Wales for help with

arrays. This work was supported by an Australian Research Council

Grant (to B. J. M.).

Conflict of interest None for all authors

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